Human growth hormone (HuGH) is a protein hormone secreted by human anterior pituitary gland, the precursor of which consists of 217 amino acid residues, wherein the first 26 amino acid residues compose a signal peptide, and the remaining 191 amino acid residues compose the mature molecule. There are two intra-molecular disulfide bonds (Cys79 and Cys191, Cys208 and Cys215), and the molecule is not glycosylated with a molecular weight of 22 kiloDaltons (kDa). The sequence of the HuGH is set forth in SEQ ID NO: 1 (NCBI: P01241, AAA72260; Denoto F M, et al. Human growth hormone DNA sequence and mRNA structure: possible alternative splicing. Nucleic Acids Res., 9: 3719-3730, 1981; Roskam W, et al. Molecular cloning and nucleotide sequence of the human growth hormone structural gene. Nucleic Acids Res., 7: 305-320, 1979; Martial J A, et al. Human growth hormone: Complementary DNA cloning and expression in bacteria. Science, 205: 602-607, 1979; Chen E Y, et al. The human growth hormone locus: nucleotide sequence, biology and evolution. Genomics, 4: 479-497, 1989). The primary functions of the HuGH include promoting the growth of a cell, organ or bone, and it is closely related to the anabolism of the body (Iglesias P, et al. Recombinant human growth hormone therapy in malnourished dialysis patients: a randomized controlled study. Am. J. Kidney Dis., 32(3): 454-463, 1998; Neely E K, Use and abuse of human growth hormone. Annu. Rev. Med., 45:407-410, 1994). After more than 20 years' clinical application of the recombinant human growth hormone (rHuGH) produced by recombinant DNA technology, the clinical efficacy and safety of the rHuGH have been demonstrated.
The results from many researches have indicated that, rHuGH shows significant therapeutic effects in treating dwarfism, burn, wound, bone fracture, bleeding ulcer, renal failure, AIDS, anabolic disorders, endogenous growth hormone deficiency dwarfism, Turner syndrome and adult growth hormone deficiency, and it also shows significant effect in anti-aging therapy. Currently rHuGH is the only effective drug for the treatment of dwarfism. By March 2001, the indications that have been approved by FDA to enter clinical research of rHuGH include: nitrogen retention intensive treatment of severe burn, short bowel syndrome (administered alone or in combination with glutamine), AIDS related growth arrest etc. Currently, the indications of rHuGH that have been approved for marketing include: adolescent endogenous growth hormone deficiency dwarfism, Turner syndrome related dwarfism, adolescent spontaneous or organ growth hormone deficiency dwarfism, Prader-Willi syndrome, premature growth disorder, AIDS related catabolic disorder, chronic renal failure related growth retardation and adult growth hormone deficiency etc.
Polyethylene glycol is an inert, nontoxic and biodegradable organic polymer, and is important in the fields of both biotechnology and pharmaceutics. PEG modification technique is to link PEG to an active protein via covalent bond. After polyethylene-glycolation (PEGylation), the properties of the protein can be significantly improved, e.g. the prolongation of drug metabolic half-life, the reduction of immunogenicity, the increase of safety, the improvement of therapeutic efficacy, the decrease of dosing frequency, the increase of drug solubility/water solubility, the increase of resistance against proteolysis, the facilitation of drug controlled release and so on (Inada et al. J. Bioact. and Compatible Polymers, 5, 343, 1990; Delgado, et al. Critical Reviews in Therapeutic Drug Carrier Systems, 9, 249, 1992; Katre, Advanced Drug Delivery Systems, 10, 91, 1993 and Davis et al. U.S. Pat. No. 4,179,337). It is disclosed in U.S. Pat. No. 4,179,337 that after linking PEG to a protein such as an enzyme or insulin, the immunogenicity of the protein was reduced, while simultaneously the activities of the protein were reduced as well, but at the same time the modified protein retained a certain proportion of the activities of the original unmodified protein. Such effect was also found in G-CSF (Satake-Ishikawa et al. Cell Structure and Function, 17, 157-160, 1992), IL-2 (Katre et al. Proc. Natl. Acad. Sci. USA, 84, 1487, 1987), TNF-α (Tsutsumi et al. Jpn. J Cancer Res., 85, 9, 1994), IL-6 (Inoue et al. J. Lab. Clin. Med., 124, 529, 1994) and CD4-IgG (Chamow et al. Bioconj. Chem., 5, 133, 1994).
It is disclosed in U.S. Pat. No. 5,824,784 that a PEG modifier with an aldehyde group at the end was used to obtain a PEG-G-CSF which was modified by a single PEG at a fixed site (N-terminal amino acid of the protein). PEG-NHS modifier synthesized by N-hydroxysuccinimide (NHS) activation can form an amido bond with ε-amino group of lysine in G-CSF. PEG-NHS has a high chemical activity but poor selectivity, and thus it is difficult to obtain a product modified by a single PEG at a fixed site. Comparing to a multi-PEGs modified product, mono-PEG modified product is more homogeneous and thus is beneficial for separation and purification, which therefore facilitates the quality control and ensures stability among batches in large-scale production.
Currently some kinds of PEGylated therapeutic protein drugs, such as PEGylated-adenosine deaminase (ADAGEN®, Enzon Pharmaceuticals), PEGylated L-asparaginase (ONCAPSPAR®, Enzon Pharmaceuticals), PEGylated interferon-α2b (PEGINTRON™, Schering-Plough) and PEGylated interferon-α2a (Pegasys, Roche), PEGylated granulocyte colony-stimulating factor (NEULASTA®, Amgen), have been applied clinically. The in vivo metabolism of the PEG moiety in a drug (or PEG itself) has already been clearly understood, and PEG has been proven to be a good and safe drug modifier without any adverse effect.
The PEG that can be linked to a protein drug normally need to be derivatized, so that one or two terminal groups at the ends of PEG can be chemically activated to possess a proper functional group which displays activity to, and thus can form a stable covalent bond with, at least one functional group of the drug to be linked. For example, PEG can be linked to ε-NH2 of a Lys residue within the protein peptide chain, or to α-NH2 of the N-terminal amino acid residue of the protein peptide chain. There are normally three forms of polyethylene glycols that have been used to modify a protein: a linear chain molecule (EP 0593868; Yu-Sen Wang et al. Advanced Drug Delivery Reviews, 54: 547-570, 2002; Yu-Sen Wang et al. Biochemistry, 39, 10634-10640, 2000), U-shaped branched molecule (EP 0809996) and Y-shaped branched molecule (CN1243779C, EP1496076). The Europe Patent no. EP0809996 describes the PEGylation of IFN-α.
It is generally believed in the art that, after PEG modification, the properties of most proteins will undergo the following changes: 1. the immunogenicity and antigenicity decrease; 2. the cyclic half-life is prolonged; 3. the solubility is increased; 4. the protein is tolerant to proteolysis; 5. the biological availability is increased; 6. the toxicity is decreased; 7. the thermostability and mechanical stability are increased; 8. The isoelectric point, electrophoretic behavior, and dynamic properties are changed, etc. Furthermore, one of the most important points is that PEG modification will result in the decrease of cellular activities of a protein, which is mainly due to the groups that have been introduced into the final product, including PEG and the linkages between PEG and the protein to be modified, and also related to the conditions of coupling as well as the generated side-product. Doris Brugger et al. (US Patent, Pub. No.: US 2004/0223950 A1) discloses that the modification products of interferon-α2a mono-modified by a single double stranded UPEG at one of different sites show significantly different in vitro anti-viral activities, wherein the modification product mono-modified by UPEG at site Lys31 has the highest specific activity, while the product mono-modified by UPEG at site Lys121 has the lowest specific activity, wherein the difference between both can be up to 5 times.
Li, Weihua et al. (China Patent Pub. No.: CN 1477126A) discloses a method of preparing a PEG modified growth hormone. It is preferred to perform the modification reaction of growth hormone by branched PEG (mPEGn-NHS) at pH 6.5-7.0, and the biological activity of the purified growth hormone mono-modified by branched PEG at a single site was measured using rats with the pituitary glands removed. The results demonstrate that, PEG coupled growth hormone (wherein PEG is double stranded PEG-NHS with a molecular weight of 40 kDa) has comparable weight increasing effect to the equal amount of growth hormone injected daily.